1. Field of Invention
This invention relates generally to the field of chemical separations, particularly to bioseparations and to improvements in multidimensional separations by the use of modular microfluidic systems, processes, devices and components.
2. Description of the Prior Art
“Bioseparation stands at the very center of effective biotechnology development.” (Bioseparation of Proteins by Ajit Sadana, Academic Press, 1998, p. ix).
Nothing occurring since 1998 has displaced separation technology from its central place in the biotechnology industry. Thus, improvements in bioseparation technology are likely to provide significant benefits to a broad range of endeavors within biotechnology. The systems, techniques and devices described herein are directed to improving separations of complex chemical mixtures, particularly complex mixtures occurring in biosystems, mixtures deriving from biosystems, and/or mixtures of interest in biotechnology. All such mixtures are referred to herein as “biofluids” for economy of language.
An effective separation (or “fractionation”) of a complex chemical mixture into fractions having distinct chemical and/or physical properties is almost always necessary for an identification, characterization and/or analysis of the mixture's component species. Moreover, the particular separation protocol that is employed is generally chosen such that the resulting fractions will, as far as feasible, have properties suitable for subsequent analytical, identification and/or characterization steps (e.g., mass spectrometry, gel electrophoresis, and many others). For economy of language herein, we use “separation” or “fractionation” to refer both to the actual separation or fractionation process as well as to the separation process plus any subsequent steps carried out on one or more of the fractions with a view towards obtaining the final result or information of interest.
When performing chemical analysis on mixtures of chemicals, particularly mixtures of biological samples such as human bodily fluids, several challenges typically arise and must be satisfactorily overcome in order to obtain reliable information concerning the chemical species of interest. The sample typically contains a very large number of distinct chemical species. For example, several thousand different proteins may be present in bodily fluids, cell lysates or other samples arising in proteomic or other biological research. Were such samples directly delivered to a typical state-of-the-art detection and/or analytical instrument (mass spectrometer, for example), such an instrument typically would not be able to distinguish, separate or resolve all the components. In particular, the components present in low concentrations are generally hidden under the more abundant components and are thereby not detected. The number of peaks containing biomolecules likely to be produced by the instrument will render it extremely difficult or impossible to achieve comprehensive analysis and obtain reliable information concerning the components of the mixture. Indeed, for many samples of biofluids arising in practice, the complexity of the sample can easily overwhelm the capability of the instrument, resulting in adequate separation or resolution for perhaps only about 30% of the components. For example, of all the currently known cancer biomarkers, most of which have been identified using targeted proteomics, over 48% would be missed by high-end proteomic platforms [M. Polanski, N. L. Anderson, Biomarker Insights 2006: 2, pp. 1-48, “A list of candidate cancer biomarkers for targeted proteomics”].
To analyze such complex mixtures of chemical species, it is typically found to be necessary that the sample be separated or fractionated into several different sub-samples or fractions before analysis is performed. For example, in order to perform mass spectrometry, it is advantageous that each fraction delivered to the mass spectrometer contain no more than about 500 different chemical species, although a smaller number of species in each fraction, typically about 50, would be more advantageous. Thus, separations are performed that concentrate different groups of chemical species into different fractions according to some chemical or physical property (or group of properties) shared by the chemical species. A large number of procedures exist for concentrating different chemical species into different fractions, and there is a corresponding huge literature describing the functions, properties and limitations of such procedures. A survey of many of these procedures with a particular focus on protein separation can be found in Sadana supra; Affinity Separations, A Practical Approach, Ed. P. Matejtschuk, Oxford University Press, 1997; Separation Methods in Proteomics, Ed. G. B. Smejkal and A. Lazareu, CRC Press, 2006; among many other references. The contents of these references are incorporated herein by reference for all purposes and particularly as related to the descriptions of separation and analytical procedure discussed herein where the cited references (and many others known and readily accessible to researchers in the field) provide detailed descriptions of instruments and procedures for performing chemical analysis.
“Multidimensional separation” typically refers to separation processes that subject the components to be separated to two or more largely independent separative displacements. See, for example, Giddings, “Concepts and Comparisons in Multidimensional Separation,” J. High Resolution Chromatography & Chromatography Comm., Vol. 10, pp. 319-323 (May, 1987). We use “multidimensional separation or fractionation” in a similarly broad sense herein referring generally to two or more fractionation steps performed sequentially without restriction as to the type, nature or number of distinct fractionation steps. In other words, in multidimensional methods of separation, different separation methods or systems are coupled. Thus, multidimensional separations can be enormously powerful but also enormously varied as the number of ways of coupling different separation techniques is far greater than the number of techniques available for combination.
An additional challenge in developing effective multidimensional processing relates to the intercompatibility (or lack thereof) between solutions used in different processing steps. For example, loading and elution buffers are often not compatible. That is, the elution buffers of one procedure are often not compatible with the loading buffers of a second procedure such that combining such procedures typically requires that a third step for buffer exchange be performed, complicating the processing procedure.
A further challenge to be met and overcome in the separation/analysis of complex chemical mixtures (especially those arising in connection with bioseparation and/or bioanalysis) is the wide variation of concentrations that may be present. For example, when analyzing for biomarkers in human bodily fluids, the concentration of various species of interest can vary by a factor of 106. when looking for biomarkers in human blood, serum albumen and IgG (Immunoglobulin-G) typically comprise around 95%-96% of all proteins present. In the remaining 4%-5%, a million-fold variation in concentration of various species can be found. Thus, while fractionation may reduce the number of chemical species from several thousand in the original sample to about 500 (or less) in each fraction, the large disparity of concentrations within each fraction further complicates the development of feasible separation and/or analytical procedures.
Proteomic analysis as typically arising in connection with the study of eukaryotic and prokaryotic cells, viruses, phages, etc. is often most concerned with the detection and analysis of low abundance proteins or proteins subject to post-translational modifications, as such species may be critically important signal-bearing molecules. The low concentrations of such species in the presence of other species, several orders of magnitude more abundant in the sample, further complicate the separation/analysis.
Yet further complications can arise when small samples require analysis, perhaps as small as microliters (μl). Manual transfer of such small samples can lead to unacceptable loss of material, and many analytical instruments contain “dead volumes” in which dilution occurs, further decreasing detection limits. That is, dead volume dilution leads to a requirement for additional quantities of sample such that the concentration arriving at the detection/analysis instrument meets the minimum requirements for the particular instrument. The need to be able to process small volumes of sample can arise from several sources including: 1) The need to enrich species present in low abundances and thereby improve the ability to detect them. 2) Some samples are available only in small volumes, for example, biopsies, multi-time point analysis of mouse serum, among others.
In addition, chromatographic/electrographic separations generally derive from the existence of different rates of travel through the separation apparatus for different chemical species, leading to a series of peaks as each chemical species emerges from the apparatus at a different time. Clearly, sharper emerging peaks facilitate the identification of closely related chemical species which may have similar rates of travel. That is, the sharper the peaks, the more peaks can be separated/resolved in a given amount of time. “Diffusion broadening” of the emerging peak occurs when the same chemical species traverses the separation apparatus with a range of different times (typically due to diffusive effects causing a species to take different paths through the apparatus). Thus, a broadened peak emerges from the apparatus, perhaps overlapping or obscuring nearby peaks, thereby reducing or destroying the analytical information that can be obtained. Diffusion broadening tends to be a more serious problem for smaller samples: as the peak broadens it's height decreases (peak area is constant) and the peak can disappear in the background, further exacerbating the detection and/or analysis of μl samples.
In brief, the separation/analysis of complex chemical mixtures, particularly mixtures arising in connection with bioseparations and/or bioanalysis, is generally a complex multi-step process, typically involving many procedures chosen from a large number of candidate procedures, which can often be carried out in numerous ways (sequencing of steps, choice of solvents, temperature, pH, among others). It is a formidable task to select from this staggering array of possibilities the proper separation/analysis protocol that enables accurate determination of chemical species of interest, for example, biological markers. For biological samples, it is frequently envisioned that the protocol will need to be performed many times on clinical samples derived from many different patients or from the same patient at different stages in the treatment or in the progression of the disease. Thus, it is clearly important to have an effective separation/analysis protocol capable of being performed reliably, rapidly and reproducibly, perhaps hundreds of times per day in a clinical environment with potentially serious consequences for inaccuracies.
It is also important to be able to test numerous candidate protocols to arrive at one or more protocols meeting the above criteria, or at least approaching those criteria as closely as is feasible. As noted above, multidimensional separations can be enormously powerful but also enormously complex in terms of the number of different techniques that can be interconnected in numerous ways and such techniques can, separately or in cooperation, be run under many different conditions to produce many different separation protocols. As ever more complex separations and analyses are required to meet the ever more complex needs of biological, medical, environmental, chemical and other fields, the full power of multidimensional separations will be required. Developing and implementing effective protocols can be a time-consuming and complex task.
Thus, there is a need in the art for improved systems, devices, and procedures for the separation and/or analysis of complex chemical mixtures, particularly mixtures of biological substances such as human bodily fluids.